The present invention relates to luciferase expression vectors, methods of making same and methods of use thereof.
Bioluminescent bacteria are widely found in both marine and terrestrial environments. Interestingly, all identified species of naturally occurring marine and terrestrial bioluminescent bacteria are Gram-negative. To date, at least eleven species in four Gram-negative genera have been described: Vibrio, Photobacterium, Shewanella (Altermonas) and Photorhabdus (Xenorhabdus). In all these species, the five genes responsible for bioluminescence are clustered in the lux operon (luxCDABE).
The bioluminescence (emitted blue-green light having a wavelength of about 490 nm) is thought to result from a luciferase-catalyzed oxidation of reduced flavin mononucleotide (FMNH2) and a long-chain fatty aldehyde. The luciferase enzyme is encoded by two subunits (luxAB), whereas the fatty acid reductase polypeptides responsible for the biosynthesis of the aldehyde substrate for the luminescent reaction are encoded by the three genes luxCDE. The genes encoding luciferase and the fatty acid reductase polypeptides have been cloned from the lux operons of Vibrio, Photobacterium and Photorhabdus and sequenced. In each case, the luxCDE genes flank the luxAB genes, with transcription in the order luxCDABE. Although a number of additional lux genes have been identified in each of these three bacteria, only luxAE are essential for the biosynthesis of light (reviewed by Meighen, E., (1993, The FASEB Journal 7:1016-1022 and Ulitzur, S., (1997), J. Biolumin Chemilumin 12:179-192).
Methods described in U.S. Pat. No. 5,650,135, make possible the detection of bioluminescent bacteria in a living animal without dissecting or otherwise opening the animal up (xe2x80x9cin vivo monitoringxe2x80x9d)xe2x80x94the light is detected through muscle, skin, fur and other traditionally xe2x80x9copaquexe2x80x9d tissues using a highly sensitive camera. In this context and others, it would therefore be desirable to confer bioluminescence properties on a bacterium of one""s choice, so that the bacterium could be followed with in vivo monitoring in various models of infection. In particular, it would be desirable to confer such bioluminescence properties on Gram-positive bacteria, since many bacteria pathogenic to mammals are in fact Gram-positive. For example, infections caused by Stapholococcus, a Gram-positive cocci, are ubiquitous and include, e.g., abscesses, mastitis, pneumonia, bacteremia, osteomyletis, enterocolitis and toxic shock syndrome (TSS). Another Gram-positive cocci, Streptococcus is the primary cause of pharyngeal infections (xe2x80x9cstrepxe2x80x9d throat). Gram-positive bacilli such as Anthrax and Listeria (which causes meningitis) can cause severe, and even fatal infections in humans and other mammals.
While a non-bioluminescent Gram-negative bacterium can typically be engineered to have bioluminescence properties by cloning into it a luxCDABE operon (under control of a suitable promoter) from a bioluminescent species (see, e.g., Contag, et al., U.S. Pat. No. 5,650,135), previous attempts to make bioluminescent Gram-positive bacteria have met with limited success. For example, one approach employed an expression cassette encoding a functional LuxAB fusion protein (Jacobs, M., et al., (1991) Mol. Gen. Genet. 230:251-256). In this cassette, a Gram-positive ribosome binding site (RBS) was inserted upstream of luxA, with the luxB gene cloned in frame downstream of luxA. Although this approach has been successful in generating a number of novel genera of bioluminescent Gram-positive bacteria useful for certain environmental and food safety studies (e.g., the assessment of food products for contamination by such bacteria), these bacteria are not useful for studying pathogenicity. A major reason for this limitation is that the LuxAB fusion proteins described in the prior art not stable at mammalian body temperatures, and are thus capable of catalyzing only minimal light production in bacterial cells at 37xc2x0 C.
In fact, none of the bioluminescent Gram-positive bacteria which have been published to date produce enough light in vivo to make them useful for the in vivo monitoring applications discussed above. It would therefore be desirable to have a method by which Gram-positive bacteria could be made to bioluminescence at temperatures found in mammalian host cells, and at levels of brightness suitable for monitoring in living animals. The present invention provides, inter alia, such methods, expression cassettes, and other tools useful for generating bioluminescent Gram-positive bacteria suitable for studies relating to infection and/or pathogenesis.
In one aspect, the invention includes an expression cassette comprising a polynucleotide encoding luxA, luxB, luxC, luxD and luxE gene products, wherein (a) the arrangement of coding sequences for the gene products is in the following relative order 5xe2x80x2-luxA-luxB-luxC-luxD-luxE-3xe2x80x2; (b) transcription of the polynucleotide results in a polycistronic RNA encoding all the gene products; and (c) each of the luxA, luxB, luxC, luxD and luxE gene products is expressed as an individual polypeptide. In one embodiment, the expression cassette includes a multiple-insertion site located adjacent the 5xe2x80x2 end of the luxA coding sequences. In another embodiment, the expression cassette further comprises at least one Gram-positive ribosome binding site sequence (SEQ ID NO:1) upstream of each of the polynucleotide sequences encoding each of the luxA, luxB, luxC, luxD and luxE gene products. The coding sequences of the gene products preferably encode a luciferase that is stable at 37xc2x0 C., such as the luciferase of Photorhabdus luminescens. Accordingly, the nucleotide coding sequences for the luciferase are preferably derived from such organisms. In one series of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sa1-Sa6; such as Sa2 or Sa4. In a related series of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sp1, Sp5, Sp6, Sp9, Sp16 and Sp17 (e.g., Sp16).
In another aspect, the invention includes an expression cassette comprising a polynucleotide encoding luxA, and luxB gene products, wherein (a) transcription of the polynucleotide results in a polycistronic RNA encoding both gene products, and (b) polynucleotide sequences comprising Gram-positive ribosome-binding site sequences are located adjacent the 5xe2x80x2 end of the luxA coding sequences and adjacent the 5xe2x80x2 end of the luxB coding sequences. In one embodiment, the expression cassette further comprises an insertion site 5xe2x80x2 to at least one of either the luxA or luxB coding sequences. The insertion site may, for example, further comprise a multiple-insertion site. In one embodiment, the multiple-insertion site is located 5xe2x80x2 to the luxA coding sequences. In a related embodiment, the multiple-insertion site is located 5xe2x80x2 to the luxB coding sequences. In another embodiment, the polynucleotide further encodes luxC, luxD and luxE gene products. The arrangement of the coding sequences for the lux gene products may be, for example, in the following relative order 5xe2x80x2-luxA-luxB-luxC-luxD-luxE-3xe2x80x2. Preferably, Gram-positive bacterial Shine-Dalgarno sequences are 5xe2x80x2 to all of the lux coding sequences. In one group of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sa1-Sa6, e.g., Sa2 or Sa4. In another group of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sp1, Sp5, Sp6, Sp9, Sp16 and Sp17, such as Sp16. As was described above, the coding sequences for luxA and luxB are preferably obtained from an organism with a luciferase that is stable at 37xc2x0 C., such as Photorhadus luminescens. 
In yet another aspect, the invention includes an expression cassette comprising a polynucleotide encoding luxA, luxB, and luc gene products, wherein (a) transcription of the polynucleotide results in a polycistronic RNA encoding all three gene products, and (b) polynucleotide sequences comprising Gram-positive bacterial Shine-Dalgarno sequences are located adjacent the 5xe2x80x2 end of the luxA coding sequences, adjacent the 5xe2x80x2 end of the luxB coding sequences, and adjacent the 5xe2x80x2 end of the luc coding sequences. In one embodiment, the polynucleotide further encodes luxC, luxD and luxE gene products. In another embodiment, Gram-positive bacterial Shine-Dalgarno sequences are located 5xe2x80x2 to all of the lux coding sequences or 5xe2x80x2 to luxA and luxC only. In one set of embodiments, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sa1-Sa6, e.g., Sa2 or Sa4. In a related set, transcription of the polynucleotide is mediated by a promoter contained in an Expression Enhancing Sequence selected from the group consisting of Sp1, Sp5, Sp6, Sp9, Sp16 and Sp17, e.g., Sp16. The expression cassette may further include a multiple-insertion site located adjacent the 5xe2x80x2 end of the luxA coding sequences. In a preferred embodiment, the coding sequences for luxA and luxB are obtained from Photorhadus luminescens. 
Also included in the invention is an expression cassette comprising a polynucleotide encoding an in-frame fusion of luxA and luxB gene products, wherein (a) polynucleotide sequences comprising Gram-positive Shine-Dalgarno sequences are located adjacent the 5xe2x80x2 end of the luxA coding sequences, and (b) an insertion site is located between the luxA and luxB coding sequences. The insertion site may further comprise a multiple-insertion site. In one embodiment, the polynucleotide further encodes luxC, luxD and luxE gene products. Arrangement of coding sequences for the gene products is preferably, but not necessarily, in the following relative order 5xe2x80x2-luxA-luxB-luxC-luxD-luxE-3xe2x80x2. In a preferred embodiment, Gram-positive bacterial Shine-Dalgarno sequences are 5xe2x80x2 to the luxA-luxB fusion coding sequences and all of the luxC, luxD, and luxE coding sequences.
It will be appreciated that all of the expression cassettes described above may be contained within a bacterial transposon or bacterial mini-transposon. Further, in all these cassettes, the coding sequences of the gene products may comprise codons that are optimal for expression of the gene products in a host system into which the expression cassette is to be introduced.
Also included in the invention is a method of selecting a light-producing expression cassette for use in a selected cell type. The method includes the steps of (i) preparing fragments of genomic DNA isolated from the selected cell type, and (ii) inserting the fragments into the insertion site of an expression cassette comprising, a polynucleotide encoding an in-frame fusion of luxA and luxB gene products, wherein (a) polynucleotide sequences comprising Gram-positive Shine-Dalgarno sequences are located adjacent the 5xe2x80x2 end of the luxA coding sequences, and (b) an insertion site is located between the luxA and luxB coding sequences. The expression cassette is preferably capable of expressing the gene products in the selected cell type. Step (iii) of the method is introducing the expression cassettes carrying the fragments into cells of the selected cell type, and step (iv) is screening for cells producing light, where the light production is mediated by the expression cassette. The fragments may be produced, for example, by enzymatic digestion of genomic DNA, partial digestion using a selected restriction endonuclease, or by mechanical fragmentation of genomic DNA. Transcription of the lux genes is preferably mediated by a promoter that is obtained from the selected cell type, for example, Staphylococcus, Streptococcus, Actinomyces, Lactobacillus, Corynebacterium, Mycobacterium, Clostridium, Propionibacterium, Enterococcus, or Bacillus. In one embodiment, the screening is carried out at a temperature greater than about 37xc2x0 C.
The invention further includes a luciferase expression cassette comprising: a) a polynucleotide encoding luc; and b) polynucleotide sequences comprising expression enhancing sequences (e.g., Gram-positive promoter and/or Gram-positive Shine-Dalgarno sequences) obtained from Gram-positive bacteria 5xe2x80x2 to the luc-encoding polynucleotide. The small DNA fragment comprising expression enhancing sequences is preferably between luc and the promoter.
The invention further includes a luciferase expression cassette comprising: a) a polynucleotide encoding luxY; and b) polynucleotide sequences comprising expression enhancing sequences (e.g., Gram-positive promoter and/or Gram-positive Shine-Dalgarno sequences) obtained from Gram-positive bacteria 5xe2x80x2 to the luxY-encoding polynucleotide. The small DNA fragment comprising expression enhancing sequences is preferably between luxY and the promoter.
Also included in the invention are the plasmids designated as pCMOR G+1 Sa1-6 and pCMOR G+2 Sp1, Sp5, Sp6, Sp9, Sp16 and Sp17.
In another aspect, the invention includes a shuttle vector comprising a) an expression cassette according to any of the expression cassettes described above; b) a polynucleotide encoding a selectable marker, c) a Gram-positive origin of replication; and d) a Gram-negative origin of replication.
Yet another aspect of the invention encompasses a method of screening for expression enhancing sequences that are useful in obtaining expression of luciferase in Gram-positive bacteria. The method comprises the steps of a) introducing DNA fragments from a Gram-positive bacterial genome into an expression cassette comprising (i) polynucleotides encoding luxA, luxB, luxC, luxD and luxE gene products, where the polynucleotides are in the following relative order 5xe2x80x2-luxABCDE; (ii) polynucleotide sequences comprising expression enhancing sequences obtained from Gram-positive bacteria 5xe2x80x2 to at least one of the lux-encoding polynucleotides and (iii) an insertion site 5xe2x80x2 to at least one of the lux-encoding polynucleotides; b) transforming the expression cassette of step (a) into a Gram-positive bacteria host cells; and c) determining the level of luciferase activity in the host cell, thereby identifying Gram-positive expression enhancing DNA sequences that are useful in obtaining expression of luciferase in Gram-positive bacteria.
Still another aspect of the invention includes a method of screening for expression enhancing sequences that are useful in obtaining expression of luciferase in Gram-positive bacteria. The method includes the steps of a) introducing DNA fragments from a Gram-positive bacterial genome into an expression cassette comprising (i) polynucleotides encoding luxA, luxB gene products (ii) polynucleotide sequences comprising expression enhancing sequences obtained from Gram-positive bacteria 5xe2x80x2 to at least one of the lux-encoding polynucleotides and (iii) an insertion site 5xe2x80x2 to at least one of the lux-encoding polynucleotides; b) transforming the expression cassette of step (a) into a Gram-positive bacteria host cells; and c) determining the level of luciferase activity in the host cell, thereby identifying Gram-positive expression enhancing DNA sequences that are useful in obtaining expression of luciferase in Gram-positive bacteria.
Also part of the invention is a method of screening for expression enhancing sequences that are useful in obtaining expression of luciferase in Gram-positive bacteria. The method comprises the steps of: a) introducing DNA fragments from a Gram-positive bacterial genome into an expression cassette comprising (i) a polynucleotide encoding luc; (ii) polynucleotide sequences comprising expression enhancing sequences obtained from Gram-positive bacteria 5xe2x80x2 to the luc-encoding polynucleotide and (iii) an insertion site 5xe2x80x2 to at least one of the luc-encoding polynucleotide; b) transforming the expression cassette of step (a) into a Gram-positive bacteria host cells; and c) determining the level of luciferase activity in the host cell, thereby identifying Gram-positive expression enhancing DNA sequences that are useful in obtaining expression of luciferase in Gram-positive bacteria.
In another aspect, the invention includes a method of making a luciferase expression cassette, comprising the steps of: (a) preparing polynucleotides encoding in a 5xe2x80x2-3xe2x80x2 direction luxA, luxB, luC, luxD and luxE gene products; and Gram-positive Shine-Dalgarno nucleotide sequences operably linked to one or more of the lux-encoding polynucleotides; and (b) inserting small sequences of nucleic acids between one or more of the polynucleotides encoding a lux gene product.
The present invention includes a method of making a luciferase expression cassette, comprising the steps of: (a) preparing polynucleotides encoding luxA and luxB gene products; and Gram-positive Shine-Dalgarno nucleotide sequences operably linked to one or more of the lux-encoding polynucleotides; and (b) inserting small sequences of nucleic acids between one or more of the polynucleotides encoding a lux gene product.
The invention also includes a method of making a luciferase expression cassette, comprising the steps of: (a) preparing polynucleotides encoding luc gene product; and Gram-positive Shine-Dalgarno nucleotide sequences operably linked to the luc-encoding polynucleotide; and (b) inserting small sequences of nucleic acids 5xe2x80x2 to the luc-encoding polynuclcotide.
The invention also includes a method of making a luciferase expression cassette, comprising the steps of: (a) preparing polynucleotides encoding luxY gene product; and Gram-positive Shine-Dalgarno nucleotide sequences operably linked to the luxY-encoding polynucleotide; and (b) inserting small sequences of nucleic acids 5xe2x80x2 to the luxY-encoding polynucleotide.
Also part of the invention is a method of modifying a Gram-positive organism to produce light, comprising transforming the Gram-positive organism with any of the expression cassettes described above.
In another aspect, the invention includes a method of screening an analyte for its ability to affect expression of a reporter marker, comprising: (a) transforming Gram-positive bacteria with any of the luciferase expression cassettes described above; (b) providing the analyte to the bacteria; (c) providing, if necessary, the substrate required for luciferase light production; and (d) monitoring the effect of the analyte on the ability of the Gram-positive bacteria to produce light, thereby identifying whether the analyte affects expression of the reporter in Gram-positive bacteria. In one embodiment, the substrate is aldehyde and is provided as a vapor.
Also included in the invention is a method of screening an analyte for its ability to affect expression of a reporter marker in a whole animal. The method includes the steps of (a) transforming Gram-positive bacteria with any of the luciferase expression cassettes described above; (b) introducing the bacteria into a whole animal; (c) providing the analyte to the animal; (d) providing, if necessary, the substrate required for luciferase light production; and (e) monitoring the effect of the analyte on the ability of the Gram-positive bacteria to produce light, thereby identifying whether the analyte affects expression of the reporter in Gram-positive bacteria. In one embodiment, the substrate is aldehyde and is provided by injection.
In another aspect, the invention includes Gram-positive bacteria capable of producing light, wherein (a) the bacteria comprise lux and luxB coding sequences, and (b) about 1xc3x97106 bacterial cells can produce at least about 1xc3x97104 Relative Light Units at about 37xc2x0 C. In other embodiments, cells emitting at least about 10 photons per second per cell are disclosed. Cells emitting at least about 25 photons per second per cell are also included. Cells emitting at least about 50 photons per second per cell are disclosed. Cells emitting at least about 75 photons per second per cell are disclosed. Cells emitting at least about 100 photons are also disclosed.
In yet another aspect, the invention includes a transgenic non-human animal comprising any of the expression cassettes described above.
Also included in the invention is a promoter sequences contained in any of Expression-enhancing sequences Sa1-Sa6 or Sp sequences (as disclosed below). In a preferred embodiment, the promoter sequence is selected from Expression-Enhancing Sequences selected from the group consisting of SEQ ID NOS:15-26.
In a general embodiment, the invention includes an expression cassette comprising a promoter sequence as defined in the above paragraph operably linked to a polynucleotide sequence encoding a light-generating protein (LGP). In one embodiment, the LGP is a fluorescent protein, such as green fluorescent protein. In another embodiment, the LGP is a luminescent or bioluminescent protein, such as luciferase. In specific embodiments, the luciferase may either a prokaryotic luciferase (a lux-encoded luciferase) or a eukaryotic (luc-encoded) luciferase.
In yet another aspect, the invention includes a method for localizing an entity in a non-human mammalian subject, comprising the following steps: (a) administering to the subject a conjugate of the entity and a prokaryotic luciferase comprising the alpha and beta subunits, (b) delivering aldehyde to the subject, (c) after a period of time in which the conjugate can achieve localization in the subject, measuring through opaque tissue, photon emission from the luciferase localized in the subject, with a photodetector device until an image of photon emission can be constructed, and (d) constructing an image of photon emission, wherein the image shows the localization of the entity in the mammalian subject.
The invention also includes bacterial host cells, for example gram-positive bacteria, comprising one or more the expression vectors, plasmids, transposons, etc described herein.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein. Furthermore, various forms of the different embodiments described herein may be combined.